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The goal of surgery for patients with newly diagnosed glioblastoma (GBM) is maximum safe resection of the contrast-enhancing (CE) lesion on magnetic resonance imaging. However, there is no consensus on the efficacy of FLAIRectomy, which is defined as the possible resection of fluid-attenuated inversion recovery (FLAIR)-hyperintense lesions surrounding the CE lesion. Although retrospective analyses suggested the potential benefits of FLAIRectomy, such outcomes have not been confirmed by prospective studies. Therefore, we planned a multicenter, open-label, randomized controlled phase III trial to evaluate the efficacy of FLAIRectomy compared with gross total resection of CE lesions in patients with newly diagnosed GBM. The primary endpoint is overall survival. In total, 130 patients will be enrolled from 47 institutions over 5 years. This trial has been registered at the Japan Registry of Clinical Trials (study number jRCT1031230245)., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Takotsubo syndrome (TTS) is characterized by transient regional left ventricular (LV) dysfunction occurring in individuals exposed to physical or emotional stress. Various stressors are triggers for TTS in cancer patients, and anti-cancer drugs have recently been proposed as a trigger. Therefore, further studies are needed to clarify these triggers and avoid the unnecessary interruption of anti-cancer treatment., Case Summary: A 66-year-old woman presented with dyspnoea 10 days after the initiation of atezolizumab in combination with bevacizumab. She had previously received osimertinib as first-line therapy for recurrent lung cancer after primary resection and atezolizumab in combination with bevacizumab, paclitaxel, and carboplatin as second-line therapy. She was admitted due to electrocardiography abnormalities and elevated troponin I and brain natriuretic peptide levels. Echocardiography revealed circumferential severe LV hypokinesis at the mid-ventricular level, with preserved wall motion at the base and apex. Cardiac catheterization performed after the attenuation of symptoms with 20 mg of intravenous furosemide showed normal coronary arteries. Cardiac magnetic resonance imaging on Day 4 revealed increases in T 1 and T 2 values and extracellular volume fraction; however, neither myocardial infiltration of inflammatory cells or myocardial necrosis was observed in endomyocardial samples obtained on the day of her arrival. Atypical TTS was suspected, and she was treated with perindopril, bisoprolol, and spironolactone. Magnetic resonance imaging 1.5 months after the onset of TTS showed improvements in LV contractility, T 1 and T 2 values, and the extracellular volume fraction., Discussion: A more detailed understanding of the relationship between anti-cancer drugs and TTS is crucial for preventing interruptions to anti-cancer therapy., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Genome-editing tools such as the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system have become essential tools for increasing the efficiency and accuracy of plant breeding. Using such genome-editing tools on maize, one of the most important cereal crops of the world, will greatly benefit the agriculture and the mankind. Conventional genome-editing methods typically used for maize involve insertion of a Cas9-guide RNA expression cassette and a selectable marker in the genome DNA; however, using such methods, it is essential to eliminate the inserted DNA cassettes to avoid legislative concerns on gene-modified organisms. Another major hurdle for establishing an efficient and broadly applicable DNA-free genome-editing system for maize is presented by recalcitrant genotypes/cultivars, since cell/tissue culture and its subsequent regeneration into plantlets are crucial for producing transgenic and/or genome-edited maize. In this study, to establish a DNA-free genome-editing system for recalcitrant maize genotypes/cultivars, Cas9-gRNA ribonucleoproteins were directly delivered into zygotes isolated from the pollinated flowers of the maize-B73 cultivar. The zygotes successfully developed and were regenerated into genome-edited plantlets by co-culture with phytosulfokine, a peptide phytohormone. The method developed herein made it possible to obtain DNA- and selectable-marker-free genome-edited recalcitrant maize genotypes/cultivars with high efficiency. This method can advance the molecular breeding of maize and other important cereals, regardless of their recalcitrant characteristics., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Objective: This is the preliminary results of a multi-center prospective clinical trial evaluating the feasibility of the hybrid of intracavitary and interstitial brachytherapy for locally advanced cervical cancer., Methods: Patients with FIGO stage IB2, IIA2, IIB, IIIA, IIIB and IVA uterine cervical cancer pretreatment width of which was ≥5 cm measured by MRI were eligible. Protocol therapy consisted of 30-30.6 Gy in 15-17 fractions of whole pelvic radiotherapy concurrent with weekly CDDP, followed by 24 Gy in 4 fractions of hybrid of intracavitary and interstitial and pelvic radiotherapy with central shield up to 50-50.4 Gy in 25-28 fractions. The primary endpoint of phase I part was that the rate of grade ≥ 3 acute non-hematologic adverse events related to hybrid of intracavitary and interstitial would be <10%., Results: Between October 2015 and October 2019, 74 patients underwent primary registration, with 52 patients eventually proceeding to the secondary registration. The median pretreatment tumor width was 5.7 cm, and FIGO Stages were IB2 10, IIA2 2, IIB 20 and IIIB 20, respectively. The median high-risk clinical target volume D90 was 72.0 Gy (54.8-86.6 Gy, EQD2), rectum D2cc was 53.7 Gy (29.3-80.3 Gy) and bladder D2cc was 69.8 Gy (38.9-84.8 Gy). The rate of grade ≥ 3 non-hematologic adverse events related to hybrid of intracavitary and interstitial was 1.9% (1/52), and 17.3% (9/52) of patients experienced non-hematologic adverse events related to hybrid of intracavitary and interstitial of any grade. In multivariate analysis, high-risk clinical target volume ≥ 35 ml was associated with an increased risk of any grade of acute non-hematologic adverse events related to hybrid of intracavitary and interstitial (P = 0.036)., Conclusion: The feasibility and reproducibility of hybrid of intracavitary and interstitial were demonstrated from a multi-center prospective clinical trial., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We aimed to predict the minimum distance between a tumor and the gastrointestinal (GI) tract that can satisfy the dose constraint by creating simulation plans with carbon-ion (C-ion) radiotherapy (RT) and photon RT for each case assuming insertion of virtual spacers of various thicknesses. We enrolled 55 patients with a pelvic tumor adjacent to the GI tract. Virtual spacers were defined as the overlap volume between the GI tract and the volume expanded 7-17 mm from the gross tumor volume (GTV). Simulation plans (70 Gy in 35 fractions for at least 95% of the planning target volume [PTV]) were created with the lowest possible dose to the GI tract under conditions that meet the dose constraints of the PTV. We defined the minimum thickness of virtual spacers meeting D2 cc of the GI tract <50 Gy as 'MTS'. Multiple regression was used with explanatory variables to develop a model to predict MTS. We discovered that MTSs were at most 9 mm and 13 mm for C-ion RT and photon RT plans, respectively. The volume of overlap between the GI tract and a virtual spacer of 14 mm in thickness (OV14)-PTV was found to be the most important explanatory variable in the MTS prediction equation for both C-ion and photon RT plans. Multiple R2 values for the regression model were 0.571 and 0.347 for C-ion RT and photon RT plans, respectively. In conclusion, regression equations were developed to predict MTS in C-ion RT and photon RT., (© The Author(s) 2021. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

Accumulated experience supports the efficacy of allogenic haematopoietic stem cell transplantation in arresting the progression of childhood-onset cerebral form of adrenoleukodystrophy in early stages. For adulthood-onset cerebral form of adrenoleukodystrophy, however, there have been only a few reports on haematopoietic stem cell transplantation and the clinical efficacy and safety of that for adulthood-onset cerebral form of adrenoleukodystrophy remain to be established. To evaluate the clinical efficacy and safety of haematopoietic stem cell transplantation, we conducted haematopoietic stem cell transplantation on 12 patients with adolescent-/adult-onset cerebral form/cerebello-brainstem form of adrenoleukodystrophy in a single-institution-based prospective study. Through careful prospective follow-up of 45 male adrenoleukodystrophy patients, we aimed to enrol patients with adolescent-/adult-onset cerebral form/cerebello-brainstem form of adrenoleukodystrophy at early stages. Indications for haematopoietic stem cell transplantation included cerebral form of adrenoleukodystrophy or cerebello-brainstem form of adrenoleukodystrophy with Loes scores up to 13, the presence of progressively enlarging white matter lesions and/or lesions with gadolinium enhancement on brain MRI. Clinical outcomes of haematopoietic stem cell transplantation were evaluated by the survival rate as well as by serial evaluation of clinical rating scale scores and neurological and MRI findings. Clinical courses of eight patients who did not undergo haematopoietic stem cell transplantation were also evaluated for comparison of the survival rate. All the patients who underwent haematopoietic stem cell transplantation survived to date with a median follow-up period of 28.6 months (4.2-125.3 months) without fatality. Neurological findings attributable to cerebral/cerebellar/brainstem lesions became stable or partially improved in all the patients. Gadolinium-enhanced brain lesions disappeared or became obscure within 3.5 months and the white matter lesions of MRI became stable or small. The median Loes scores before haematopoietic stem cell transplantation and at the last follow-up visit were 6.0 and 5.25, respectively. Of the eight patients who did not undergo haematopoietic stem cell transplantation, six patients died 69.1 months (median period; range 16.0-104.1 months) after the onset of the cerebral/cerebellar/brainstem lesions, confirming that the survival probability was significantly higher in patients with haematopoietic stem cell transplantation compared with that in patients without haematopoietic stem cell transplantation ( P  = 0.0089). The present study showed that haematopoietic stem cell transplantation was conducted safely and arrested the inflammatory demyelination in all the patients with adolescent-/adult-onset cerebral form/cerebello-brainstem form of adrenoleukodystrophy when haematopoietic stem cell transplantation was conducted in the early stages. Further studies are warranted to optimize the procedures of haematopoietic stem cell transplantation for adolescent-/adult-onset cerebral form/cerebello-brainstem form of adrenoleukodystrophy., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain.)

In vitro fertilization (IVF) systems using isolated gametes have been utilized to dissect post-fertilization events in angiosperms, since the female gametophytes of plants are deeply embedded within ovaries. In addition, IVF systems have been used to produce hybrid and polyploid zygotes. Complete IVF systems have been established in maize and rice, two of three major crop species providing the majority of human energy intake. Among those crop species, gametes of wheat have not been used to establish a complete IVF system successfully. In this study, a wheat IVF system was developed to introduce the advantages of this technology to wheat research. Fusion of gametes was performed via a modified electrofusion method, and the fusion product, a zygote, formed a cell wall and two nucleoli. The first division of zygotes was observed 19-27 h after fusion, and the resulting two-celled embryo developed into 10-20-celled globular-like embryos and multicellular club-shaped embryos by 3 and 7-10 d after fusion, respectively. Such zygotic division profiles were mostly consistent with those in the embryo sac, suggesting that the division profile of IVF-produced early embryos reflects that of early embryos in planta. Although the IVF-produced club-shaped embryos did not develop into differentiated embryos but into compact embryonic calli, fertile plants could be regenerated from the embryonic calli, and the seeds harvested from those plants grew normally into seedlings. The IVF system described here might become an important technique for generating new genotypes of wheat and/or new hybrids as well as for investigating fertilization-induced events in wheat., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Here, we cloned the CpRLK1 gene, which encodes a receptor-like protein kinase expressed during sexual reproduction, from the heterothallic Closterium peracerosum-strigosum-littorale complex, one of the closest unicellular alga to land plants. Mating-type plus (mt(+)) cells with knockdown of CpRLK1 showed reduced competence for sexual reproduction and formed an abnormally enlarged conjugation papilla after pairing with mt(-) cells. The knockdown cells were unable to release a naked gamete, which is indispensable for zygote formation. We suggest that the CpRLK1 protein is an ancient cell wall sensor that now functions to regulate osmotic pressure in the cell to allow proper gamete release., (© The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Root hairs are fast-growing tubular protrusions on root epidermal cells that play important roles in water and nutrient uptake in plants. The tip-focused polarized growth of root hairs is accomplished by the secretion of newly synthesized materials to the tip via the polarized membrane trafficking mechanism. Here, we report the function of two different types of plasma membrane (PM) Qa-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), SYP123 and SYP132, in the growth of root hair in Arabidopsis. We found that SYP123, but not SYP132, localizes in the tip region of root hairs by recycling between the brefeldin A (BFA)-sensitive endosomes and the PM of the expanding tip in an F-actin-dependent manner. The vesicle-associated membrane proteins VAMP721/722/724 also exhibited tip-focused localization in root hairs and formed ternary SNARE complexes with both SYP123 and SYP132. These results demonstrate that SYP123 and SYP132 act in a coordinated fashion to mediate tip-focused membrane trafficking for root hair tip growth.

Multidisciplinary cancer boards (CBs) for making cancer treatment decisions have become popular in many countries; however, the status of radiotherapy in CBs and the influence of CBs on radiotherapy decisions have not been studied. To clarify these issues, we reviewed the minutes of our CBs from February 2010 to March 2012, and we classified planned treatments discussed at the CBs into five categories and analyzed decisions concerning radiotherapy in each category. The fraction of cases for which radiotherapy was recommended was 536/757 (71%). These cases included 478 cases (63%) for which radiation therapy was planned and four cases (0.5%) for which radiation therapy was unexpectedly recommended. On the other hand, radiation therapy was canceled in 21 cases (4%) for which radiation therapy had been planned. This study showed that radiotherapy was discussed in many cases at CBs and that CBs have a great influence on decisions concerning radiotherapy.

While most studies regarding reactive oxygen species (ROS) focus on their deleterious biological effects, a growing body of evidence indicates the importance of ROS as critical mediators of several signaling pathways, including those involved in hematopoiesis. In this study, we show the critical role of ROS in lineage decision of myeloid progenitors. In megakaryocyte-erythrocyte progenitor cells (MEP), intracellular ROS levels were found to be as low as those in hematopoietic stem cells (HSC). In contrast, remarkably high intracellular ROS levels were observed in granulocyte-monocyte progenitor cells. Intracellular ROS levels in common myeloid progenitors (CMP) were inversely correlated with their MEP differentiation potential. Moreover, gene set enrichment analysis revealed that ROS-low CMP showed gene expression patterns similar to those of MEP, indicating that intracellular ROS levels mark the fate of CMP. In in vitro assays, ROS significantly suppressed the generation of MEP and the formation of megakaryocyte-erythrocyte colonies from CMP. In ROS-high CMP, expression of colony-stimulating factor one receptor (CSF1R) was highly upregulated, and its surface expression correlated with their granulocyte-monocyte differentiation potential. Furthermore, ROS was found to induce the expression of CSF1R mRNA in a leukemia cell line. These data provide novel insights into the relationship between ROS and the hematopoietic differentiation system., (© AlphaMed Press.)

We evaluated whether reduced-dose chemotherapy with 2 days of idarubicin (12 mg/m(2)) and 5 days of cytarabine (100 mg/m(2)) (2 + 5) is effective for patients aged 65-74 by retrospectively comparing the results with those aged 55-64 treated with 3 + 7. In 1999-2009, we treated 20 patients aged 65-74 with 2 + 5, and 23 patients aged 55-64 with 3 + 7. The complete remission rates by the first induction were 50.0 and 69.6% for older and younger groups (P = 0.203). Two-year overall survival rates were 55.9 and 32.3% for older and younger groups; 2-year rates of relapse-free survival for all these patients were 15.7 and 36.5%. The differences in overall and relapse-free survival were statistically insignificant (P = 0.726 and 0.413, respectively). The treatment results of 2 + 5 for the older group were not significantly worse compared with those of 3 + 7 for the younger. Therefore, elderly patients who do not tolerate 3 + 7 should still benefit from 2 + 5.

We report a 19-year-old patient with systemic-onset juvenile idiopathic arthritis (JIA) who developed a mediastinal germinoma during treatment with infliximab. Although the cancer risk of infliximab is controversial, this agent may have accelerated the growth of the germinoma. We conclude that the indications for tumor necrosis factor (TNF) inhibitors should be strictly decided and that a nationwide cohort study is necessary to assess the risk of cancer in patients with JIA exposed to biologics.

Understanding the mechanisms of DNA compaction is becoming increasingly important for gene therapy and nanotechnology DNA applications. The kinetics of the compaction velocity of single DNA molecules was studied using two non-protein condensation systems, poly(ethylene glycol) (PEG) with Mg(2+) for the polymer-salt-induced condensation system and spermine for the polyamine condensation system. The compaction velocities of single tandem λ-DNA molecules were measured at various PEG and spermine concentrations by video fluorescent microscopy. Single DNA molecules were observed using a molecular stretching technique in the microfluidic flow. The results show that the compaction velocity of a single DNA molecule was proportional to the PEG or spermine concentration to the power of a half. Theoretical considerations indicate that the compaction velocity is related to differences in the free energy of a single DNA molecule between the random coil and compacted states. In the compaction kinetics with PEG, acceleration of the compaction velocity occurred above the overlap concentration while considerable deceleration occurred during the coexistence state of the random coil and the compacted conformation. This study demonstrates the control factors of DNA compaction kinetics and contributes toward the understanding of the compaction mechanisms of non-protein DNA interactions as well as DNA-protein interactions in vivo.

Background: Although influenza virus resistance to the neuraminidase inhibitor zanamivir is reported less frequently than is resistance to the neuraminidase inhibitor oseltamivir in clinical settings, it is unknown whether this difference is due to the limited use of zanamivir or to an inherent property of the drug. We therefore compared the prevalence of drug-resistant viruses and virus shedding in seasonal influenza virus-infected children treated with either oseltamivir or zanamivir., Methods: Clinical specimens (throat or nasal swab) were collected from a total of 144 pediatric influenza patients during the 2005-2006, 2006-2007, 2007-2008, and 2008-2009 influenza seasons. Neuraminidase inhibitor-resistant mutants were detected among the isolated viruses by sequencing the viral hemagglutinin and neuraminidase genes. Sensitivity of the viruses to neuraminidase inhibitors was tested by neuraminidase inhibition assay., Results: In oseltamivir- or zanamivir-treated influenza patients who were statistically comparable in their age distribution, vaccination history, and type or subtype of virus isolates, the virus-shedding period in zanamivir-treated patients was significantly shorter than that in oseltamivir-treated patients. Furthermore, the frequency of zanamivir-resistant viruses was significantly lower than that of oseltamivir-resistant viruses., Conclusion: In comparison with treatment with oseltamivir, treatment of pediatric patients with zanamivir resulted in the emergence of fewer drug-resistant influenza viruses and a shorter virus-shedding period. We conclude that zanamivir shows promise as a better therapy for pediatric influenza patients.

Recently, two small-molecule kinase inhibitors targeting epidermal growth factor receptor have proven effective in the treatment of non-small cell lung cancer. There are specific activating mutations within the tyrosine kinase domain of epidermal growth factor receptor related to the sensitivity of tyrosine kinase inhibitors. However, it is unknown whether rare mutations in the N-lobe (exons 18-20) and the C-lobe (exon 21) of the epidermal growth factor receptor kinase domain other than L858R in exon 21 and the in-frame deletion in exon 19 may predict the effectiveness of epidermal growth factor receptor-tyrosine kinase inhibitors. We reported a case of non-small cell lung cancer harboring a rare epidermal growth factor somatic mutation, codon 768 AGC > ATC in exon 20 (S768I), who showed a good clinical response to gefitinib. Therefore, we may suggest that this rare mutation (S768I in exon 20) may not be an insensitive epidermal growth factor receptor somatic mutation in vivo.

Rapid and multiplex detection system using an ultrasensitive DNA microarray was developed and utilized for the analysis of six pharmacokinetically relevant single nucleotide polymorphisms (SNPs) (MDR1-C1236T, MDR1-G2677TA, MDR1-C3435T, CYP3A5-A6986G, CYP2C19-G681A, CYP2C19-G636A) from blood samples derived from liver transplant patients. The SNP detection system is comprised of three processes: multiplex PCR, single base extension with fluorescently labelled di-deoxy-nucleotides and detection by DNA microarray. The entire workflow of this system completes within 5 h. The final genotype call was obtained statistically by Mahalanobis distance which was calculated from the bi-coloured fluorescent signals detected by the microarray. In order to detect the six SNPs, this system required only 50 copies of genomic DNA, and the obtained detection calls completely matched with the results by the sequencing-based genotyping method. With the high sensitivity and rapid processing, our SNP detection system utilizing ultrasensitive microarray is a promising device applicable for diagnostic utility.

Japanese toads (Bufo japonicus) migrate to and from breeding sites in the early spring, possibly guided by olfactory cues. We previously showed that the electrical activity of olfactory receptor neurons (ORNs) in the toads was enhanced in the breeding period. We undertook morphological and physiological studies of the olfactory epithelium to determine whether any cellular substrate of the epithelium underlies the enhanced electrical activity of ORNs. The ORNs of the toads were labeled by antiserum to olfactory marker protein (OMP), and the morphology of the labeled cells and their distribution in the epithelium were examined throughout the year. The OMP-positive cells, distributed mainly in the basal and intermediate layers of the epithelium, were most numerous in the early breeding period. Cell proliferation in the epithelium detected by 5-bromo-2'-deoxyuridine labeling was most elevated in this period. The electrical activity of ORNs was examined by recording the electroolfactogram (EOG) in the toads throughout the year. Statistical analysis showed a positive correlation between the density of OMP-positive cells in the epithelium and the amplitude of the EOG responses. A greater number of ORNs in the breeding period possibly aids the toads in migrating to their breeding sites.

The nasal cavity and olfactory bulb (OB) of the Japanese jungle crow (Corvus macrorhynchos) were studied using computed tomography (CT) and histochemical staining. The nasal septum divided the nasal cavity in half. The anterior and maxillary conchae were present on both sides of the nasal cavity, but the posterior concha was indistinct. A small OB was present on the ventral surface of the periphery of the cerebrum. The OB-brain ratio--the ratio of the size of the OB to that of the cerebral hemisphere--was 6.13. The olfactory nerve bundles projected independently to the OB, which appeared fused on gross examination. Histochemical analysis confirmed the fusion of all OB layers. Using a neural tracer, we found that the olfactory nerve bundles independently projected to the olfactory nerve layer (ONL) and glomerular layer (GL) of the left and right halves of the fused OB. Only 4 of 21 lectins bound to the ONL and GL. Thus, compared with mammals and other birds, the jungle crow may have a poorly developed olfactory system and an inferior sense of olfaction. However, it has been contended recently that the olfactory abilities of birds cannot be judged from anatomical findings alone. Our results indicate that the olfactory system of the jungle crow is an interesting research model to evaluate the development and functions of vertebrate olfactory systems.

Membrane trafficking to the plasma membrane (PM) is a highly organized process which enables plant cells to build up their bodies. SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) genes, which encode the proteins involved in membrane trafficking, are much more abundant in the Arabidopsis genome than in that of any other eukaryote. We have previously shown that a large number of SNARE molecules in the Arabidopsis cell are localized predominantly on the PM. In the present study, in order to elucidate the physiological function of each PM-localized SNARE, we analyzed the spatiotemporal expression profiling of nine SYP1s that are resident in the PM of Arabidopsis, and used the information thus acquired to generate transgenic Arabidopsis plants expressing green fluorescent protein-fused Qa-SNAREs under control of their authentic promoters. Among the nine SYP1s, only SYP132 is expressed ubiquitously in all tissues throughout plant development. The expression patterns of the other SYP1s, in contrast, are tissue specific, and all different from one another. A particularly noteworthy example is SYP123, which is predominantly expressed in root hair cells during root development, and shows a focal accumulation pattern at the tip region of root hairs. These results suggest that SYP132 is involved in constitutive membrane trafficking to the PM throughout plant development, while the other SYP1s are involved in membrane trafficking events such as root formation or tip growth of root hair, with some redundancy.

Transmission electron microscopy was used to study the responses of the supporting cells of the olfactory epithelium at 1-5 days after surgical ablation of the olfactory bulb (bulbectomy). In intact olfactory epithelium, lamellar smooth endoplasmic reticulum and rod-shaped mitochondria were distinctly observed in the supporting cells. On the first day after bulbectomy, bending of the microvilli and an increase in the smooth endoplasmic reticulum were observed. Cristae of the mitochondria became obscure, and the density of the mitochondrial matrix decreased. On the second day after bulbectomy, the number of microvilli decreased, broad cytoplasmic projections that contained cytoplasmic organelles protruded into the luminal side, and the mitochondria were swollen. On the fifth day after bulbectomy, microvilli seemed to be normal and some cells had large cytoplasmic projections that protruded toward the lumen of the nasal cavity. Within the cytoplasmic projections of the supporting cells, a large lamellar and reticular-shaped smooth endoplasmic reticulum was evident. Mitochondria exhibited almost normal morphology. The current findings demonstrate that morphological changes occur in the supporting cells after bulbectomy. This new evidence hypothesizes that these changes represent events that contribute to the regeneration of the olfactory epithelium after bulbectomy.

Background: We compared the clinical effectiveness of oseltamivir and zanamivir in children with influenza A (H1N1) virus, influenza A (H3N2) virus, and influenza B virus infections., Methods: Total febrile period and the duration of fever after the start of treatment were compared between an oseltamivir-treated group (mean age, 8.9 years; range, 4.0-15.9 years) and a zanamivir-treated group (mean age, 10.0 years; range, 4.0-15.7 years) in the pediatric outpatient clinics of our hospitals. Oseltamivir was used to treat 91 children with influenza A (H3N2) infection and 24 children with influenza A (H1N1) infection. Zanamivir was used to treat 35 children with influenza A (H3N2) infection and 12 children with influenza A (H1N1) infection. Oseltamivir was also used to treat 128 children with influenza B virus infection, and zanamivir was used to treat 59 with influenza B virus infection., Results: There was no statistically significant difference in total febrile period or duration of fever after the start of treatment between the oseltamivir-treated group and the zanamivir-treated group of children with influenza A (H3N2) infection (mean duration of febrile period, 2.40 days vs. 2.39 days; mean duration of fever after the start of treatment, 1.35 days vs. 1.40 days), influenza A (H1N1) (mean duration of febrile period, 2.60 days vs. 2.46 days; mean duration of fever after the start of treatment, 1.79 days vs, 1.54 days), or influenza B (mean duration of febrile period, 2.95 days vs. 2.84 days; mean duration of fever after the start of treatment, 1.86 days vs. 1.67 days). Oseltamivir was more effective against influenza A (H3N2) than against influenza A (H1N1) or influenza B., Conclusions: Oseltamivir and zanamivir were equally effective in reducing the febrile period of children with influenza A (H1N1), influenza A (H3N2), and influenza B virus infection.

To date, over 100 vomeronasal receptor type 1 (V1R) genes have been identified in rodents. V1R is specifically expressed in the rodent vomeronasal organ (VNO) and is thought to be responsible for pheromone reception. Recently, 21 putatively functional V1R genes were identified in the genome database of the amphibian Xenopus tropicalis. Amphibians are the first vertebrates to possess a VNO. In order to determine at which point during evolution the vertebrate V1R genes began to function in the vomeronasal system, we analyzed the expression of all putatively functional V1R genes in Xenopus olfactory organs. We found that V1R expression was not detected in the VNO but was specifically detected in the main olfactory epithelium (MOE). We also observed that V1R-expressing cells in the MOE coexpressed Gi2, thus suggesting that the V1R-Gi2-mediated signal transduction pathway, which is considered to play an important role in pheromone reception in the rodent VNO, exists in the amphibian MOE. These results suggest that V1R-mediated signal transduction pathway functions in Xenopus main olfactory system.

In mammalian brown adipose tissue, uncoupling protein 1 (UCP1), an integral inner mitochondrial membrane protein, triggers a proton leak and converts the energy generated by the resulting electron flow into heat. Although the recent finding of plant UCPs in non-thermogenic tissues has questioned their involvement in thermogenesis, there are few studies of plant UCPs in thermogenic tissues. Therefore, in this work, two cloned UCP cDNAs, SrUCPA and SrUCPB, isolated from the thermogenic spadix of skunk cabbage, were analysed. SrUCPA, not SrUCPB, was identified as the major uncoupling protein, and it was found to be integrated into the inner mitochondrial membrane. Topological analyses indicate that the 1st and 2nd intra-matrix loops are sensitive to trypsin treatment, but the 3rd intra-matrix loop is resistant to it. Using spadix mitochondria, the uncoupling activity of SrUCPA was examined. Although SrUCPA transcripts were constitutively expressed in various tissues irrespective of thermogenic stage, the SrUCPA protein was detected only in the thermogenic tissue or stage. On the other hand, both gene and protein expression for another heat-generating protein, SrAOX, were increased specifically in the thermogenic tissue or stage. Quantitative immunoblot analysis revealed that SrUCPA was an abundant protein in spadix mitochondria, accounting for about 3% of the total mitochondrial protein in the spadix. The results suggest that specific co-expression of SrUCPA and SrAOX protein in the thermogenic tissue or stage, as well as the high expression of SrUCPA protein in spadix mitochondria, may play a role in thermogenesis of skunk cabbage.

Dihydrosphingosine C4 hydroxylase is a key enzyme in the biosynthesis of phytosphingosine, a major constituent of sphingolipids in plants and yeasts. The rice genome contains five homologue genes for dihydrosphingosine C4 hydroxylase, DSH1-DSH5, whose gene products show high degrees of homology to the yeast counterpart, SUR2. Among them, expression of DSH1, DSH2 and DSH4 was detected, and DSH1 and DSH4 complement the yeast sur2 mutation. The DSH1 gene was specifically and abundantly expressed in vascular bundles and apical meristems. In particular, very strong expression was detected in the stigmas of flowers. Repression of DSH1 expression by the antisense gene or RNA interference (RNAi) resulted in a severe reduction of fertility. In the transformants in which DSH1 expression was suppressed, significantly increased expression of DSH2 was found in leaves but not in pistils, suggesting that there was tissue-specific correlation between DSH1 and DSH2 expression. Our results indicate that the product of DSH1 may be involved in plant viability or reproductive processes, and that the phenotype of sterility is apparently caused by loss of function of DSH1 in the stigma. It is also suggested that there is a complex mechanism controlling the tissue-specific expression of the DSH1 gene.

Background: Recently, many Japanese physicians have claimed that oseltamivir is less effective in children with influenza B virus infection. This study assesses the effectiveness of oseltamivir against influenza A (H3N2) and influenza B in children on the basis of the duration of febrile illness., Methods: We used oseltamivir to treat 127 children with influenza A (H3N2; mean age, 6.97 years [range, 1-15 years]) and 362 children with influenza B (mean age, 5.16 years [range, 1-15 years]) in outpatient clinics. The duration of fever after the start of oseltamivir therapy was compared in the influenza A group and the influenza B group., Results: The mean duration of fever after the start of oseltamivir therapy was significantly greater in the influenza B group than in the influenza A (H3N2) group (2.18 days vs. 1.31 days, respectively; P<.001). The difference was marked in young children (1-5 years old; 2.37 days for the influenza B group vs. 1.42 days for the influenza A group) but was not significant among older children (11-15 years old). The 50% inhibitory concentration of oseltamivir against influenza B virus was 75.4+/-41.7 nmol/L and was substantially higher than that for type A (H3N2) virus (0.3+/-0.1 nmol/L). Only 3 (1.6%) of 192 influenza B viruses were resistant to oseltamivir., Conclusions: Oseltamivir is much less effective against influenza B virus infection in young children, probably because of the low sensitivity of influenza B viruses to oseltamivir. The effectiveness of oseltamivir against influenza B is influenced by age and host immunity. A few oseltamivir-resistant influenza B strains were isolated before the start of oseltamivir therapy.

Previously, a coculture system of accessory olfactory bulb (AOB) neurons and vomeronasal (VN) neurons was established for studying the functional roles of AOB neurons in pheromonal signal processing. In this study, the effect of VN neurons on the development of AOB neurons was examined in a coculture system. Spine density was quantitatively measured for various culture periods of 21, 28, 36, and 42 days in vitro. The densities of dendritic spines were lower in the coculture than in single culture for all periods in vitro. Synapse formation on spines was analyzed immunocytochemically using an anti-synaptophysin antibody. The ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. The volume of spine head was larger in the coculture than in single culture. These changes were not observed in the coculture in which there was no physical contact between AOB neurons and VN neurons. These observations suggest that synapse formation on the spines of AOB neurons is modified by physical contact with VN neurons.

We have developed a new DNA chip whose substrate has a unique minute columnar array structure made of plastic. The DNA chip exhibits ultrahigh sensitivity, up to 100-fold higher than that of reference DNA chips, which makes it possible to monitor gene expression profiles even with very small amounts of RNA (0.1-0.01 microg of total RNA) without amplification. Differential expression ratios obtained with the new DNA chip were validated against those obtained with quantitative real-time PCR assays. This novel microarray technology would be a powerful tool for monitoring gene expression profiles, especially for clinical diagnosis.

Methods to predict the responsiveness of a particular tumor to a particular anticancer drug are desirable not only for chemotherapy but also for chemoradiotherapy. Here, we examined the effects of viral or activated oncogenes on sensitivity to anticancer drugs by using SHOK (Syrian hamster Osaka-Kanazawa) cells and their transfectants. The IC50 of each transfectant was compared with that of the pSV2Neo transfected control. Cells transfected with the c-myc, v-mos, or v-fgr gene increased their sensitivity to bleomycin, while those transfected with the H-ras gene developed resistance. Resistance to cisplatin was conferred by the introduction of the H-ras or c-cot gene. In the case of adriamycin, the c-myc or c-cot transfectant increased sensitivity and the H-ras transfectant decreased it. Mitomycin C resistance was observed by the introduction of the K-ras gene. Thus, the H-ras gene was found to be involved in the development of resistance to three of the four anticancer drugs. In addition, we have for the first time shown that mos and cot have an effect on sensitivity to three and all of the four anticancer drugs, respectively. These results suggest that the expression of each oncogene would differently affect sensitivity to the four anticancer drugs used in this study, and this property could be a possible marker to predict chemosensitivity.

To analyze the mechanisms of perception and processing of pheromonal signals in vitro, we previously developed a new culture system for vomeronasal receptor neurons (VRNs), referred to as the vomeronasal pocket (VN pocket). However, very few VRNs were found to express the olfactory marker protein (OMP) and to have protruding microvilli in VN pockets, indicating that these VRNs are immature and that VN pockets are not appropriate for pheromonal recognition. To induce VRN maturation in VN pockets, we here attempted to coculture VN pockets with a VRN target-accessory olfactory bulb (AOB) neurons. At 3 weeks of coculture with AOB neurons, the number of OMP-immunopositive VRNs increased. By electron microscopy, the development of microvilli in VRNs was found to occur coincidentally with OMP expression in vitro. These results indicate that VRN maturation is induced by coculture with AOB neurons. The OMP expression of VRNs was induced not only by AOB neurons but also by neurons of other parts of the central nervous system (CNS). Thus, VRN maturation requires only CNS neurons. Since the maturation of VRNs was not induced in one-well separate cultures, the nonspecific induction of OMP expression by CNS neurons suggests the involvement of a direct contact effect with CNS in VRN maturation.

Recent findings suggest that macrophage-tropic human immunodeficiency virus type 1 (HIV-1) produced in colostrum/early breast milk may hold a clue to determine the mechanisms of transmission of HIV-1 via breast-feeding. Here, we show that the majority of CD4(+) cells in the colostrum are CD14(+) macrophages expressing both chemokine receptors and DC-SIGN, a dendritic cell-specific receptor for HIV-1. The R5-type macrophage-tropic HIV-1 isolate NL(AD8) infected such breast-milk macrophages and caused them to secrete virus particles efficiently; however, the secreted virions showed only a weak transmissibility to their susceptible target, MAGIC-5 cells. When stimulated with interleukin-4, the breast-milk macrophages demonstrated a striking enhancement of expression of DC-SIGN and showed a strong capacity to transmit NL(AD8) virions to MAGIC-5 cells, which was specifically blocked by anti-DC-SIGN-specific antibody. These results suggest that HIV-1 virions captured by DC-SIGN, but not secreted cell-free virions, may be more efficiently transmitted to other compartments, such as the gastrointestinal tract, through acidic gastric juice.

The vomeronasal (VN) systems of rodents and opossums are of the segregated type, i.e alpha-subtype G protein Gi2- or Go-expressing VN neurons, which are sensory cells, project discretely to the rostral or caudal region of the accessory olfactory bulb (AOB). Although this zone-specific projection is believed to be a common feature for processing pheromones in mammals, we previously found a uniform-type VN system in goat in which only Gi2-expressing VN axons terminate at the AOB. In most mammals, it remains unclear whether their VN systems are of the segregated or uniform type. Therefore, we investigated morphologically the VN systems of different mammalian species (dog, horse, musk shrew and common marmoset). Consequently, all VN axons of the examined animals were positively stained with immunohistochemistry for Gi2 in the same way as that in the goat. On the other hand, we observed immunoreactivities against Go in the olfactory axons, but not in the VN axons. These results suggest that many mammals have uniform-type VN systems, and at least two types of VN systems exist in terrestrial mammals. This morphological evidence will help us determine the processing function of VN systems.

Phosphoinositide 3-kinases (PI3Ks) constitute a family of lipid kinases that regulate an array of fundamental cellular responses by neutrophils [polymorphonuclear leukocytes (PMN)]. p85alpha Gene-disrupted mice were used to help accurately identify the physiological role of the PI3K isoform in PMN activation in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF). PMN from the p85alpha-/- mice showed normal cellular motility, and the quantity of superoxide anion (O(2(-))) produced by PMN upon stimulation with formyl-Met-Leu-Phe did not significantly differ between p85alpha-/- and wild-type mice under controlled conditions. In p85alpha-/- mice, the O(2(-)) production by PMN was enhanced (primed) by GM-CSF when stimulated with the chemotactic peptide but to a significantly lesser extent than in wild-type mice. In addition, no major GM-CSF-dependent delay in apoptosis or activation of Akt protein phosphorylation by GM-CSF was observed in the p85alpha-/- mice. In terms of targeting strategy, however, the mutation actually expressed a small amount of Ia-type (p85alpha-regulated) PI3K activity (partially abrogated) in the mice. These results demonstrate that Ia-type PI3K plays a critical role in the enhancement of the GM-CSF-modulated function of PMN and in the PI3K/Akt pathway-dependent delay of PMN apoptosis.

PTB-like protein (PTBLP) is a new homologue of pyrimidine tract binding protein (PTB), and has been cloned as a possible autoantigen in cancer-associated retinopathy. PTBLP has two functional domains, the nuclear localization signal and the RNA recognition motifs (RRMs). Full-length PTBLP (PTBLP-L) has four RRMs, and its alternative splicing product (PTBLP-S) lacks the third and fourth RRMs. Although PTBLPs are expressed in neuronal tissues, the function of PTBLPs has not been determined. We have studed whether PTBLP plays a role in neuronal differentiation using PC12 cells. During the process of nerve growth factor-induced neuronal differentiation of PC12 cells, PTBLP-L was down-regulated whereas PTBLP-S was up-regulated. Transfection of PTBLP-L into PC12 cells led to the suppression of neuronal differentiation. In PTBLP-S transfected cells, however, this suppression was not evident. When both PTBLP-L and PTBLP-S were co-transfected, the suppressive effect of PTBLP-L decreased. In differentiated cells, PTBLP-S localized in the nucleus and PTBLP-L was found dispersed throughout the cytoplasm and neuronal growth cone. These findings suggest that PTBLP-L acts as a negative regulator of neuronal differentiation and PTBLP-S acts as a competitor of PTBLP-L.

Mammals possess two anatomically and functionally distinct olfactory systems. The olfactory epithelium (OE) detects volatile odorants, while the vomeronasal organ (VNO) detects pheromones that elicit innate reproductive and social behavior within a species. In rodent VNO, three multigene families that encode the putative pheromone receptors, V1Rs, V2Rs and V3Rs, have been expressed. We have identified the V1R homologue genes from goat genomic DNA (gV1R genes). Deduced amino acid sequences of gV1R genes show 40-50% and 20-25% identity to those of rat and mouse V1R and V3R genes, respectively, suggesting that the newly isolated goat receptor genes are members of the V1R gene family. One gene (gV1R1 gene) has an open reading frame that encodes a polypeptide of 309 amino acids. It is expressed not only in VNO but also in OE. In situ hybridization analysis revealed that gV1R1 -expressing cells were localized in neuropithelial layers of VNO and OE. These results suggest that the goat may detect pheromone molecules through two distinct olfactory organs.

To detect the biological activity of mammalian putative pheromone receptors (V1Rs and V2Rs), the mouse V1R gene was introduced into a primary culture of vomeronasal cells using the adenovirus expression system, and the response of these cells to mouse urine was analyzed by calcium imaging. These cells specifically responded to male but not female mouse urine. This response was attenuated by pertussis toxin, a specific inhibitor of G-protein G(ialpha)/G(oalpha) coupling from receptors. Our findings indicate that a putative pheromone receptor was specifically activated by mouse urine, a major source of mouse pheromones, and suggest that G(i)/G(o) are functionally coupled with the receptor.

To investigate in detail the distribution of G protein subtypes G(i)2alpha and G(o)alpha along the surface of the vomeronasal epithelium, we used double labeling immunocytochemical methods and electron microscopy. We examined the immunoreactivity of these surface structures with antibodies against G(i)2alpha and G(o)alpha. G(i)2alpha- and G(o)alpha-positive cells were observed at the epithelial surface and were evenly distributed. Electron microscopy revealed that strong immunoreactivities to both antibodies were observed on the microvilli and knob-like surface structures of receptor cells. No immunoreactivity was found on the microvilli or surface membranes of supporting cells. This expression pattern is similar to that reported for putative pheromone receptors. These data confirm that there are two distinct classes of vomeronasal receptor cells expressed at the surface of the epithelium. These two classes of receptors correspond to the same G(i)2alpha- and G(o)alpha-positive cells distributed in cell body layers of the epithelium and in the axon terminals in the accessory olfactory bulb.

Cancer-associated retinopathy (CAR) is a rare form of retinal degeneration and also one of the paraneoplastic neurologic disorders. Sera of CAR patients usually contain high titers of antibodies against retinal proteins, and CAR is believed to be an autoimmune disease. Using serum from a CAR patient as a molecular probe, a homologue of the polypyrimidine tract-binding protein (PTB) was isolated from a cDNA library of rat neonatal retina. This homologue, named PTB-like protein (PTBLP), encodes a 532 amino acid residue protein and has 73.5 and 68.8% homology with PTB and with a regulator of differentiation 1, respectively. Functional domains in the PTB, such as nuclear localization signals and four RNA recognition motifs (RRMs), were highly conserved. The expression of PTBLP mRNA was observed in the retina and brain but not in liver, kidney, spleen, or lung. The expression of PTBLP protein in rat retina was distributed in most of the cells in the ganglion cell layer and some cells in the inner nuclear layer. The PTBLP protein was localized in the nuclei of these cells. These results suggest that PTBLP is a new member of the PTB gene family and a neuron-specific homologue.

Goats have a well-developed vomeronasal (VN) system and exhibit pheromone-induced reproductive facilitation, but there are no reports on the projection pattern of VN neurons in this species. Rodent, guinea pig and opossum accessory olfactory bulbs (AOBs) have been shown to have a segregated pattern of projection of the VN neurons, which express the two alpha-subtypes of the G-protein, namely Gi2 and Go, to the rostral and caudal regions of the AOB, respectively. In this study we investigated the projection pattern of VN nerve terminals by immunocytochemical staining of the goat vomeronasal organ (VNO) and the AOB with antibodies to Gi2 and Go. Gi2-immunoreactivity was found on the luminal surface of the sensory epithelium of the VNO, and in the VN nerve and glomerular layer throughout the AOB. On the other hand, Go-immunoreactivity was not identified in either the VNO or the VN nerve layer of the AOB. These results indicate that the projection pattern of VN neurons from the VNO to the AOB in the goat is considerably different from that in rodents which show a distinct segregated pattern.

The distribution of sugar chains on tissue sections of the rat vomeronasal epithelium, and the adhesive force between the sugar and its specific lectin were examined with an atomic force microscope (AFM). AFM tips were modified with a lectin, Vicia villosa agglutinin, which recognizes terminal N-acetyl-D-galactosamine (GalNAc). When a modified tip scanned the luminal surface of the sensory epithelium, adhesive interactions between the tip and the sample surface were observed. The final rupture force was calculated to be approximately 50 pN based on the spring constant of the AFM cantilever. Distribution patterns of sugar chains obtained from the force mapping image were very similar to those observed using fluorescence-labeled lectin staining. AFM also revealed distribution patterns of sugar chains at a higher resolution than those obtained with fluorescence microscopy. Most of the adhesive interactions disappeared when the scanning solution contained 1 mM GaINAc. The adhesive interactions were restored by removing the sugar from the solution. Findings suggest that the adhesion force observed are related to the binding force between the lectin and the sugars distributed across the vomeronasal epithelium.

Recent studies suggest a protective effect of glucocorticoid against progression of bone erosion and periarticular osteoporosis in patients with rheumatoid arthritis (RA), although this steroid hormone itself is believed to increase bone loss. To understand the antagonistic effect of glucocorticoid for osteopenic process in RA patients, we examined the effect of dexamethasone on Fas-mediated apoptosis of cultured human osteoblasts induced by either anti-Fas IgM or activated peripheral blood mononuclear cells (PBMC). Human osteoblastic cell line MG63 and primary osteoblast-like cells obtained from biopsy specimens were used in this study. PBMC isolated from healthy donors were cultured with or without recombinant interleukin-2 (rIL-2) followed by 12-O-tetradecanoyl-phorbol 13-acetate (PMA) with ionomycin in the presence or absence of dexamethasone. Fas was functionally expressed on MG63 and primary osteoblast-like cells, and treatment of these cells with dexamethasone affected neither Fas expression nor anti-Fas IgM-induced apoptosis. Activated PBMC expressing membrane-type Fas ligand (mFasL) efficiently killed both MG63 and primary osteoblasts-like cells, and the addition of human Fas chimeric protein (hFas-Fc) significantly diminished the cytotoxicity, indicating that interactions between mFasL of activated PBMC and Fas on human osteoblasts induce apoptosis of the latter. Although dexamethasone did not affect apoptosis of MG63 and primary osteoblast-like cells induced by anti-Fas IgM, treatment of activated PBMC with dexamethasone markedly inhibited both mFasL expression and cytotoxicity of these cells against human osteoblasts, suggesting that dexamethasone preferentially acts not on osteoblasts but PBMC. Cultured supernatants from activated PBMC induced apoptosis of human osteoblasts and the addition of hFas-Fc also inhibited the cytotoxicity of the supernatants. In addition, soluble form FasL (sFasL) was detected in the supernatants of activated PBMC. Furthermore, both the cytotoxicity and sFasL concentration of cultured supernatants of activated PBMC incubated with dexamethasone was significantly lower than that in the absence of dexamethasone. Our data suggest that glucocorticoid suppresses the apoptotic process of osteoblasts by inhibiting the expression of both mFasL and sFasL derived from activated PBMC, mediating a protective effect against periarticular bone loss and bone erosion in inflammatory arthritis such as RA.

Chemoreceptor cells in the vomeronasal and olfactory epithelium are replaced following experimentally induced degeneration. This study analyzes quantitatively the time course and degree of vomeronasal receptor cell replacement. Unilateral transection of the vomeronasal nerves in adult hamster was used to induce a retrograde degeneration of receptor cells in the vomeronasal organ. Histological measurement of both number of receptor cells and epithelial thickness were made for recovery times from 0 to 60 days. After nerve transection, there was a gradual degeneration of receptor cells, the number decreasing to 50% of control by day 2 and 16% by day 6. During days 7-15 maximum receptor cell replacement was observed. Cell number increased rapidly and reached a peak on day 15. At recovery times of 40-60 days, cell number returned to the control level. Epithelial thickness, however, decreased to 60-70% during the degeneration period (days 4-6) and did not return to control levels. After 40-60 days epithelial thickness remained at 70% of control. These results demonstrate that vomeronasal receptor cells are replaced following degeneration, but epithelial thickness does not return to control levels. These findings suggest that the number of replacement cells is not limited by the reduced thickness of the epithelium, and that recovery mechanisms may function to restore an optimum number of receptor cells.

The present study was designed to determine whether excitatory amino acids directly act on the gonadotropin-releasing hormone (GnRH) nerve terminals to release the peptide. The median eminence taken from ovariectomized rats was dual immunostained with GnRH and ionotropic glutamate receptor subtypes (NR1, GIuR1, GluR2/3, GluR6/7 and KA2), and their colocalization was examined under electron microscopy. The connection of fibers immunopositive for GnRH and glutamate was also examined. Of the glutamate receptor subtypes, NR1- and KA2-immunoreactivities were colocalized with GnRH-immunoreactivity in nerve terminals of the median eminence. In addition, some glutamate-immunopositive nerve terminals were shown to abut the many GnRH-immunopositive nerve terminals. No synaptic contacts were observed on these immunopositive nerve terminals. These results suggest that GnRH release is regulated at the GnRH nerve terminals by excitatory amino acids in a non-synaptic manner in the median eminence.

Simultaneous brain microdialysis in tumour and non-tumour tissues has been used for kinetic determination of the local distribution of an anticancer agent, cisplatin, in rats. Rat brain was implanted with 9L malignant glioma and cisplatin (3.5 mg kg-1) was administered as a selective intracarotid infusion for 30 min to rats prepared for brain microdialysis. The amount of platinum in the dialysate collected from tumour and non-tumour brain tissues was determined by atomic absorption spectrophotometry, as representative of cisplatin. Total and free platinum concentrations in plasma were also measured. Free platinum is accumulated preferentially in the tumour tissue and the brain tumour distribution coefficient (the ratio of brain tumour platinum AUC to plasma free platinum AUC, where AUC is the area under the platinum concentration-time curve) was 0.69, although there was little distribution into normal brain tissue. Drug binding to plasma proteins was 65%. It is concluded that simultaneous microdialysis is an easy and available method for assessing in-vivo local pharmacokinetics and distribution of cisplatin in tumour and non-tumour tissues of the brain.

The purpose of this study is to investigate the ocular membrane permeability and the permeation mechanism of hydrophilic drugs such as thyrotropin-releasing hormone (TRH), p-nitrophenyl beta-cellopentaoside (PNP) and luteinizing hormone-releasing hormone (LHRH). The penetration of hydrophilic drugs was measured across the isolated corneal and conjunctival membranes of albino rabbits using a two-chamber diffusion glass cell. The corneal permeabilities of hydrophilic drugs were much lower than those of beta blockers reported previously. The corneal penetration of TRH was the highest among the hydrophilic drugs studied. Scraping the corneal epithelium increased the penetration of hydrophilic drugs. Conjunctival membranes showed higher permeability to hydrophilic drugs compared with corneal membranes. The permeability of drugs was also analysed by Fick's equation. The partition parameter and diffusion parameter of TRH, PNP and LHRH in the cornea were lower than those in scraped cornea and conjunctiva. In addition to the data of fluorescein isothiocyanate-dextran reported previously, the permeability coefficient of hydrophilic drugs through the cornea, scraped cornea and conjunctiva correlated with molecular weight of the drugs. The diffusion parameters of hydrophilic drugs decreased with an increase of molecular weight for all ocular membranes. The extent of dependency of partition parameters on the molecular weights of drugs varied according to the ocular membrane. These results indicate that ocular membranes are sufficiently different in permeation character and mechanism to control the extent and pathway for ocular absorption of hydrophilic drugs.

Ocular membranes have been characterized by in-situ absorption of the ophthalmic beta-blockers carteolol (hydrophilic) and timolol and befunolol (lipophilic) using a cylindrical cell. After introduction of drug solution into the cell on the cornea, sclera (bulbar conjunctival and scleral layer) or palpebral conjunctiva, the disappearance of the drug from the cell was determined as in-situ absorption. The ophthalmic drugs disappeared from the conjunctival and scleral membranes although disappearance from the cornea was hardly observed. The conjunctival membrane showed the highest permeability. Lipophilic drugs were more permeable than hydrophilic. In-situ apparent permeability coefficients of the ophthalmic drugs through the conjunctiva and sclera correlated with the lipophilicity of drugs. A high drug concentration in the aqueous humor was observed after corneal application. There is a relationship between concentration in the aqueous humor was observed after corneal application. There is a relationship between concentrations of drugs in the aqueous humor and previously reported in-vitro apparent permeability coefficients of the drugs in the cornea. This in-situ method using a cylindrical cell is a useful method of investigating the ocular absorption of ophthalmic drugs.

The purpose of this study was to evaluate the effect of absorption enhancer on in-vivo transdermal absorption of cyclosporin using intradermal microdialysis in rats. Cyclosporin oily solutions (0.5, 2, 8% w/v) were prepared from Sandimmun (10% w/v oily oral preparation of cyclosporin) by diluting with olive oil. 1-[2-(Decylthio)ethyl] azacyclopentan-2-one (HPE-101) and glycerin were added to the cyclosporin formulation as an absorption enhancer at various concentrations between 1 and 20%. These formulations were applied to the shaved abdomen of rats treated with intradermal microdialysis at a flow rate of 2.5 microL min-1 for 6 h. Cyclosporin was immediately detected and attained a plateau in the dermal dialysate after topical application of cyclosporin oily solution alone. Cyclosporin levels in the dialysate increased with increasing cyclosporin concentrations in the formulation from 0.5 to 8% (w/v). HPE-101 did not influence cyclosporin absorption at concentrations less than 6% (w/v). Addition of 10% (w/v) HPE-101 significantly enhanced an apparent absorption rate of cyclosporin by 4.9 times. However, 20% (w/v) HPE-101 did not show the enhancing activity. On the other hand, addition of glycerin at concentrations of 6, 10, and 20% (v/v) significantly enhanced an apparent absorption rate of cyclosporin by 3.0, 6.4, and 6.9 times, respectively. The time lag for cyclosporin absorption was less than 0.21 h in all tested cases. This microdialysis study shows that glycerin is a suitable enhancer for improving the in-vivo cyclosporin absorption from the skin.